Adenylosuccinate synthetase and method for producing purine nucleotides using the same

ABSTRACT

The present disclosure relates to an adenylosuccinate synthetase variant, a microorganism containing the same, and a method for preparing purine nucleotides using the microorganism.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 16/482,615 filed Jul. 31, 2019, now pending, which is a U.S.national phase application of PCT/KR2018/009714, filed Aug. 23, 2018,which claims priority to KR Application No. 10-2018-0089855, filed Aug.1, 2018. U.S. application Ser. No. 16/482,615 is herein incorporated byreference in its entity.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 200187_446D1_SEQUENCE_LISTING.txt. The text fileis 19 KB, was created on Dec. 13, 2021, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present disclosure relates to a novel adenylosuccinate synthetase, amicroorganism containing the same, and a method for preparing purinenucleotides using the microorganism.

BACKGROUND ART

5′-Inosine monophosphate (hereinafter, IMP), a nucleic acid-basedmaterial, is an intermediate of the nucleic acid biosynthetic metabolicsystem used in various fields (e.g., medicines, various medicalapplications, etc.), and is a material widely used as a food seasoningadditive or food along with 5′-guanine monophosphate (hereinafter, GMP).It is known that IMP itself produces a beef flavor and enhances theflavor of monosodium glutamic acid (MSG) like GMP, thus attractingpublic attention as a taste-based nucleic acid-based seasoning.

Methods of preparing IMP may include a method of enzymatically degradingribonucleic acid extracted from yeast cells, a method of chemicallyphosphorylating inosine produced by fermentation (Agri. Biol. Chem., 36,1511(1972), etc.), a method of culturing a microorganism that directlyproduces IMP and recovering IMP from the cultured medium, etc. Amongthese methods, the most widely used method is that of using amicroorganism capable of directly producing IMP.

Additionally, the method of preparing GMP may include a method ofconverting xanthosine 5′-monophosphate (hereinafter, XMP) produced bymicrobial fermentation into GMP using a coryneform microorganism and amethod of converting XMP produced by microbial fermentation into GMPusing Escherichia coli. In the above methods, when GMP is produced by amethod where XMP is produced first and then converted into GMP, theproductivity of XMP (i.e., a precursor of the conversion reaction duringthe microbial fermentation) must be enhanced, and additionally, both theproduced XMP and the GMP already produced during the entire process ofthe conversion reaction should be protected from being lost.

Meanwhile, since enzymes in nature do not always exhibit optimalproperties in terms of activity, stability, substrate specificity tooptical isomers, etc. in industrial applications, various attempts havebeen made to improve enzymes to achieve the desired use by variation oftheir amino acid sequences. Among these, rational design andsite-directed mutagenesis of enzymes have been applied to improvefunctions of enzymes in some cases; however, these methods havedisadvantages in that information on the structure of the target enzymeis not sufficient or the structure-function correlation is not clear,and thus they cannot be effectively applied. In this case, it has beenreported that the activity of an enzyme can be enhanced by improving theenzyme through a directed evolution method, in which enzymes of thedesired traits are screened from a mutant library of enzymes constructedthrough random variations of enzyme genes. The inventors of the presentdisclosure have performed extensive research for high-yield productionof purine nucleotides by a method producing purine nucleotidescontaining IMP or XMP through the microbial fermentation. As a result,they have discovered protein variants having higher productivity ofpurine nucleotides, thereby completing the present disclosure.

DISCLOSURE Technical Problem

An object of the present disclosure is to provide an adenylosuccinatesynthetase variant.

Another object of the present disclosure is to provide a polynucleotideencoding the adenylosuccinate synthetase variant.

Still another object of the present disclosure is to provide a vectorcontaining the polynucleotide.

Still another object of the present disclosure is to provide amicroorganism capable of producing purine nucleotides, which containsthe adenylosuccinate synthetase variant and the vector.

Still another object of the present disclosure is to provide a methodfor preparing purine nucleotides, which includes culturing themicroorganism of the genus Corynebacterium in a medium; and recoveringthe purine nucleotides from the microorganism or the medium.

Technical Solution

Hereinbelow, exemplary embodiments of the present disclosure will bedescribed in detail. Meanwhile, each of the explanations and exemplaryembodiments disclosed herein can be applied to other explanations andexemplary embodiments. That is, all of the combinations of variousfactors disclosed herein belong to the scope of the present disclosure.Furthermore, the scope of the present disclosure should not be limitedby the specific disclosure provided hereinbelow.

To achieve the above objects, an aspect of the present disclosureprovides an adenylosuccinate synthetase variant in which the 85^(th)amino acid from the N-terminus of the amino acid sequence of SEQ ID NO:2 is substituted with a different amino acid. The modifiedadenylosuccinate synthetase has a modification on the amino acid at the85^(th) position from the N-terminus of the amino acid sequence of SEQID NO: 2. Specifically, the present disclosure provides anadenylosuccinate synthetase variant having at least one amino acidvariation in the amino acid sequence of SEQ ID NO: 2, in which themodification includes a substitution of the 85^(th) position from theN-terminus with a different amino acid.

As used herein, the term “adenylosuccinate synthetase” refers to anenzyme having an important role in purine biosynthesis. For the purposeof the present disclosure, the enzyme refers to a protein involved inthe production of purine nucleotides including 5′-inosine monophosphate(IMP) or 5′-xanthosine monophosphate (XMP).

In the present disclosure, SEQ ID NO: 2 refers to an amino acid sequencehaving the activity of adenylosuccinate synthetase. Specifically, SEQ IDNO: 2 is a protein sequence having the activity of adenylosuccinatesynthetase encoded by purA gene. The amino acid sequence of SEQ ID NO: 2may be obtained from NCBI GenBank, which is a known database. In anembodiment, the amino acid sequence of SEQ ID NO: 2 may be derived froma microorganism of the genus Corynebacterium, but is not limitedthereto, and may include any sequence having the same activity as theabove amino acid sequence without limitation. Additionally, the scope ofthe amino acid sequence of SEQ ID NO: 2 may include the amino acidsequence of SEQ ID NO: 2 having the activity of adenylosuccinatesynthetase or an amino acid sequence having 80% or more homology oridentity to the amino acid sequence of SEQ ID NO: 2, but is not limitedthereto. Specifically, the above amino acid sequence may include theamino acid sequence of SEQ ID NO: 2 and/or an amino acid sequence havingat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology oridentity to the amino acid sequence of SEQ ID NO: 2. The amino acidsequence having homology or identity may be those in the above range,excluding a sequence having 100% identity, or may be a sequence havingless than 100% identity. Additionally, it is apparent that any proteinhaving an amino acid sequence having deletion, modification,substitution, or addition in part of the sequence can also be used inthe present disclosure as long as it has the homology or identity andexhibits efficacy corresponding to that of the above protein.

In the present disclosure, the term “adenylosuccinate synthetasevariant” may be used interchangeably with a polypeptide variant havingpurine nucleotide productivity, a purine nucleotide-producing variantpolypeptide, a polypeptide variant producing purine nucleotides, apolypeptide variant having the adenylosuccinate synthetase activity, anadenylosuccinate synthetase variant, etc. Additionally, the protein maybe derived from the genus Corynebacterium stationis, but the protein isnot limited thereto.

The adenylosuccinate synthetase variant includes a modification of theamino acid at the 85^(th) position from the N-terminus in the amino acidsequence of SEQ ID NO: 2. The adenylosuccinate synthetase variant isthat where the 85^(th) amino acid in the amino acid sequence of SEQ IDNO: 2 is substituted with a different amino acid. The adenylosuccinatesynthetase variant may include the amino acid sequence of SEQ ID NO: 2or it may be an adenylosuccinate synthetase variant having weakeractivity compared to a non-variant adenylosuccinate synthetase derivedfrom a wild-type microorganism. Such an adenylosuccinate synthetasevariant indicates the modification of the 85^(th) amino acid from theN-terminus in the amino acid sequence of SEQ ID NO: 2 or the amino acidsequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% ormore homology or identity to the amino acid sequence of SEQ ID NO: 2, asexplained above.

Specifically, the adenylosuccinate synthetase variant is that where the85^(th) amino acid in the amino acid sequence of SEQ ID NO: 2 issubstituted with serine, alanine, valine, leucine, methionine,isoleucine, threonine, asparagine, glutamine, cysteine, tyrosine,lysine, aspartic acid, or glutamic acid, and the adenylosuccinatesynthetase variant may have weaker activity of adenylosuccinatesynthetase compared to that of a polypeptide including the amino acidsequence of SEQ ID NO: 2, but the adenylosuccinate synthetase variant isnot limited thereto.

For the purpose of the present disclosure, when a microorganism includesthe adenylosuccinate synthetase variant, the amount of purine nucleotideproduction including IMP or XMP is increased. This is meaningful in thatthe present disclosure enables the increase of the amount of IMP or XMPproduction through the adenylosuccinate synthetase variant of thepresent disclosure while the wild-type Corynebacterium strain cannotproduce IMP or XMP, or can only produce a very small amount even if IMPor XMP is produced.

The adenylosuccinate synthetase variant may include an amino acidsequence selected from the group of amino acid sequences where the85^(th) amino acid from the N-terminus in the amino acid sequence of SEQID NO: 2 is substituted with an amino acid selected from the groupconsisting of serine, alanine, valine, leucine, methionine, isoleucine,threonine, asparagine, glutamine, cysteine, tyrosine, lysine, asparticacid, and glutamic acid.

Specifically, the adenylosuccinate synthetase variant may be comprisedof a polypeptide including an amino acid sequence, which is selectedfrom the group of amino acid sequences where the 85^(th) amino acid fromthe N-terminus in the amino acid sequence of SEQ ID NO: 2 is substitutedwith an amino acid selected from the group consisting of serine,alanine, valine, leucine, methionine, isoleucine, threonine, asparagine,glutamine, cysteine, tyrosine, lysine, aspartic acid, and glutamic acid.Additionally, the adenylosuccinate synthetase variant may include anamino acid sequence where the 85^(th) amino acid from the N-terminus inthe amino acid sequence of SEQ ID NO: 2 is substituted with a differentamino acid, which has the amino acid sequence of the adenylosuccinatesynthetase variant or an amino acid sequence having 80% or more homologyor identity to the amino acid sequence of the adenylosuccinatesynthetase variant, but the amino acid sequence is not limited thereto.Specifically, the adenylosuccinate synthetase variant of the presentdisclosure may include a polypeptide having at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% or more homology or identity to the amino acidsequence, where the 85^(th) amino acid in the amino acid sequence of SEQID NO: 2 is substituted with an amino acid selected from the groupconsisting of serine, alanine, valine, leucine, methionine, isoleucine,threonine, asparagine, glutamine, cysteine, tyrosine, lysine, asparticacid, and glutamic acid. Additionally, it is apparent that any aminoacid sequence having the above sequence homology or identity andexhibiting an effect corresponding to that of the protein must alsobelong to the scope of the present disclosure, even if part of the aminoacid sequence may have deletion, modification, substitution, or additionin part of the sequence, in addition to the amino acid at the 85^(th)position.

That is, although the present disclosure describes “protein orpolypeptide having the amino acid sequence of a particular SEQ ID NO”,it is apparent that a protein having an amino acid sequence withdeletion, modification, substitution, or addition in part of thesequence can also be used in the present disclosure, as long as theprotein has activity identical or corresponding to that of thepolypeptide comprised of an amino acid sequence of the corresponding SEQID NO. For example, as long as a protein has the activity identical orcorresponding to that of the polypeptide variant, it does not excludesequence addition, naturally occurring mutation, silent mutation, orconservative substitution thereof which does not alter the functions ofthe protein, before and after the amino acid sequence. It is apparentthat a protein having such a sequence addition or mutation also fallswithin the scope of the present disclosure.

The “conservative substitution” means replacement of an amino acid withanother amino acid having similar structural and/or chemical properties.Such an amino acid substitution may generally occur based on similarityin polarity of residues, charge, solubility, hydrophobicity,hydrophilicity, and/or amphipathic nature. For example, positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include glutamic acid andaspartic acid; aromatic amino acids include phenylalanine, tryptophan,and tyrosine; and hydrophobic amino acids include alanine, valine,isoleucine, leucine, methionine, phenylalanine, tyrosine, andtryptophan.

Accordingly, in the present disclosure, the “variant” may furtherinclude conservative substitution and/or modification of at least oneamino acid in the “protein or polypeptide having an amino acid sequenceof a particular SEQ ID NO”. For example, certain variants may includevariants in which at least one part, such as a N-terminal leadersequence or transmembrane domain, is removed. Other variants may includevariants in which a part is removed from the N-terminus and/orC-terminus of a mature protein. The variant may also include othermodifications, including deletion or addition of amino acids, which haveminimal effects on the properties and a secondary structure of thepolypeptide. For example, the polypeptide may be conjugated to a signal(or leader) sequence at the N-terminus of a protein thatco-translationally or post-translationally directs transfer of aprotein. The polypeptide may also be conjugated to another sequence or alinker to facilitate identification, purification, or synthesis of thepolypeptide. The term “variant” may be used interchangeably withmodification, modified protein, modified polypeptide, mutant, mutein,divergent, etc., and any term may be used without limitation, as long asit is used in a sense of being modified.

Homology and identity mean a degree of relatedness between two givenamino acid sequences or nucleotide sequences and may be expressed as apercentage.

The terms “homology” and “identity” may often be used interchangeablywith each other.

Sequence homology or identity of a conserved polynucleotide orpolypeptide may be determined by a standard alignment algorithm anddefault gap penalties established by a program to be used may be used incombination. Substantially, homologous or identical sequences mayhybridize under moderately or highly stringent conditions along theirentire sequence or at least about 50%, about 60%, about 70%, about 80%,or about 90% of the entire length. With regard to the polynucleotides tobe hybridized, polynucleotides including a degenerate codon instead of acodon may also be considered.

Whether any two polynucleotide or polypeptide sequences have homology,similarity, or identity may be determined by, for example, a knowncomputer algorithm such as the “FASTA” program using default parametersas in Pearson et al. (1988) (Proc. Natl. Acad. Sci. USA 85]: 2444).Alternatively, they may be determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443 to 453) asperformed in the Needleman program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276 to 277) (version 5.0.0 or later) (including GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12: 387(1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] et al., J Molec Biol215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,]Academic Press, San Diego, 1994, and [CARILLO ETA/.](1988) SIAM JApplied Math 48: 1073). For example, homology, similarity, or identitymay be determined using BLAST or ClustalW of the National Center forBiotechnology Information.

Homology, similarity, or identity of polynucleotides or polypeptides maybe determined by comparing sequence information using a GAP computerprogram (e.g., Needleman et al. (1970), J Mol Biol 48: 443) as disclosedin Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAPprogram defines similarity as the number of aligned symbols (i.e.,nucleotides or amino acids) which are similar, divided by the totalnumber of symbols in the shorter of the two sequences. The defaultparameters for the GAP program may include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)and the weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix) of Gribskov et al. (1986) Nucl. Acids Res.14: 6745, as disclosed by Schwartz and Dayhoff, eds., Atlas Of ProteinSequence And Structure, National Biomedical Research Foundation, pp. 353to 358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10penalty for each symbol in each gap (or gap open penalty 10, gapextension penalty 0.5); and (3) no penalty for end gaps. Therefore, theterm “homology” or “identity”, as used herein, represents relevancebetween sequences.

Additionally, it is apparent that a polynucleotide which can betranslated, due to codon degeneracy, into a polypeptide variantcomprised of an amino acid sequence where the 85^(th) amino acid fromthe N-terminus of the amino acid sequence of SEQ ID NO: 2 is substitutedwith a different amino acid, or a polypeptide variant having homology oridentity thereto may also be included. Additionally, by hybridizationunder stringent conditions with a probe that can be prepared from aknown gene sequence (e.g., a sequence complementary to all or part ofthe nucleotide sequence), any polynucleotide sequence encoding anadenylosuccinate synthetase variant including an amino acid sequence,where the 85^(th) amino acid of the amino acid sequence of SEQ ID NO: 2is substituted with an amino acid selected from the group consisting ofserine, alanine, valine, leucine, methionine, isoleucine, threonine,asparagine, glutamine, cysteine, tyrosine, lysine, aspartic acid, andglutamic acid, may be included without limitation.

Another aspect of the present disclosure relates to a polynucleotideencoding the adenylosuccinate synthetase variant, or a vector includingthe polynucleotide.

As used herein, the term “polynucleotide” refers to a DNA or RNA strandhaving more than a certain length as a nucleotide polymer, which is along chain of nucleotide monomers connected by covalent bonds, and morespecifically, to a polynucleotide fragment encoding the polypeptidevariant.

The polynucleotide encoding the polypeptide variant of the presentdisclosure may include any polynucleotide sequence without limitation,as long as it encodes the polypeptide variant having the activity ofadenylosuccinate synthetase. In the present disclosure, the geneencoding the amino acid sequence of adenylosuccinate synthetase is purAgene, and specifically, the gene may be derived from Corynebacteriumstationis, but is not limited thereto.

Specifically, due to codon degeneracy or by considering codons preferredby a microorganism in which the polypeptide is able to be expressed,various modifications may be made in the coding region of thepolynucleotide within the scope that does not change the amino acidsequence of the polypeptide. Any polynucleotide sequence may be includedwithout limitation as long as it encodes the adenylosuccinate synthetasevariant, where the 85^(th) amino acid in the amino acid sequence of SEQID NO: 2 is substituted with a different amino acid.

Additionally, by hybridization under stringent conditions with a probethat can be prepared from a known gene sequence (e.g., a sequencecomplementary to all or part of the nucleotide sequence), any sequenceencoding a protein having the activity of an adenylosuccinate synthetasevariant, where the 85^(th) amino acid in the amino acid sequence of SEQID NO: 2 is substituted with a different amino acid, may be includedwithout limitation.

The “stringent conditions” refer to conditions that enable specifichybridization between polynucleotides. Such conditions are described indetail in the literature (e.g., J. Sambrook et al., supra). Thestringent conditions may include conditions under which genes havinghigh homology or identity (e.g., genes having 40% or more, specifically90% or more, more specifically 95% or more, still more specifically 97%or more, particularly specifically 99% or more homology or identity) canhybridize to each other; conditions under which genes having lowerhomology or identity cannot hybridize to each other; or conditions whichare common washing conditions for Southern hybridization (e.g., a saltconcentration and a temperature corresponding to 60° C., 1×SSC, 0.1%SDS; specifically 60° C., 0.1×SSC, 0.1% SDS; more specifically 68° C.,0.1×SSC, 0.1% SDS, once, specifically, twice or three times).

Hybridization requires that two nucleic acids have complementarysequences, although mismatches between bases may be possible dependingon hybridization stringency. The term “complementary” is used todescribe the relationship between nucleotide bases that can hybridize toeach another. For example, with respect to DNA, adenosine iscomplementary to thymine and cytosine is complementary to guanine.Accordingly, the present disclosure may also include isolated nucleicacid fragments complementary to the entire sequence as well as tosubstantially similar nucleic acid sequences.

Specifically, a polynucleotide having homology or identity may bedetected using hybridization conditions including a hybridization stepat T_(m) of 55° C. and by utilizing the above-described conditions.Additionally, the T_(m) value may be 60° C., 63° C., or 65° C., but isnot limited thereto, and may be appropriately controlled by thoseskilled in the art according to the purpose.

The appropriate stringency for hybridizing polynucleotides depends onthe length of the polynucleotides and the degree of complementation, andvariables are well known in the art (see Sambrook et al., supra, 9.50 to9.51, 11.7 to 11.8).

In the present disclosure, the gene encoding the amino acid sequence ofthe adenylosuccinate synthetase variant is purA gene, and thepolynucleotide encoding the gene is the same as explained above.

In the present disclosure, the polynucleotide encoding theadenylosuccinate synthetase variant is also the same as explained above.

As used herein, the term “vector” refers to a DNA construct containingthe nucleotide sequence of the polynucleotide encoding the targetpolypeptide which is operably linked to an appropriate control sequencesuch that the target polypeptide is expressed in an appropriate host.The control sequence may include a promoter to initiate transcription,any operator sequence to control such transcription, a sequence encodingan appropriate ribosome-binding site on mRNA, and a sequence to controltermination of transcription and translation. Upon transformation intoan appropriate host, the vector may replicate or function independentlyof the host genome, or may integrate into the genome itself.

The vector used in the present disclosure may not be particularlylimited as long as the vector is replicable in the host cell, and anyvector known in the art may be used. Examples of the vector commonlyused may include natural or recombinant plasmids, cosmids, viruses, andbacteriophages. For example, as a phage vector or cosmid vector, pWE15,M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc.may be used, and as a plasmid vector, those based on pBR, pUC,pBluescriptII, pGEM, pTZ, pCL, pET, etc. may be used. Specifically, pDZ,pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BACvector, etc. may be used.

In an embodiment, the polynucleotide encoding the target polypeptide maybe inserted into the chromosome through a vector for chromosomalinsertion. The insertion of the polynucleotide into the chromosome maybe performed using any method known in the art (e.g., by homologousrecombination), but the method is not limited thereto. A selectionmarker for confirming the insertion of the vector into the chromosomemay be further included. The selection marker was used for selection ofcells transformed with the vector (i.e., for confirmation of presence ofthe insertion of the target nucleic acid molecule), and markers capableof providing selectable phenotypes (e.g., drug resistance, auxotrophy,resistance to cytotoxic agents, and expression of surface polypeptides)may be used. Under the circumstances where selective agents are treated,only the cells capable of expressing the selection markers can surviveor express other phenotypic traits, and thus the transformed cells canbe easily selected.

Still another aspect of the present disclosure provides a microorganismproducing purine nucleotides by containing the adenylosuccinatesynthetase variant or a polynucleotide encoding the adenylosuccinatesynthetase variant. Specifically, the microorganism containing theadenylosuccinate synthetase variant and/or the polynucleotide encodingthe adenylosuccinate synthetase variant may be a microorganism preparedby transformation using a vector containing the polynucleotide, but themicroorganism is not limited thereto.

As used herein, the term “transformation” refers to a process ofintroducing a vector which includes a polynucleotide encoding a targetprotein into a host cell such that the protein encoded by thepolynucleotide can be expressed in the host cell. It does not matterwhether the transformed polynucleotide is inserted into the chromosomeof the host cell and located thereon or located outside of thechromosome, as long as the transformed polynucleotide can be expressedin the host cell. Further, the polynucleotide may include DNA and RNAencoding the target protein. The polynucleotide may be introduced in anyform, as long as the polynucleotide can be introduced into the host celland expressed therein. For example, the polynucleotide may be introducedinto the host cell in the form of an expression cassette, which is agene construct including all elements required for its autonomousexpression. The expression cassette may include a promoter, atranscription termination signal, a ribosome binding site, and atranslation termination signal that may be operably linked to thepolynucleotide. The expression cassette may be in a form of anexpression vector performing self-replication. In addition, thepolynucleotide may be introduced into the host cell as is to be operablylinked to the sequence required for expression in the host cell, but isnot limited thereto.

Additionally, the term “operably linked” refers to a functional linkagebetween the gene sequence and a promoter sequence which initiates andmediates transcription of the polynucleotide encoding the targetpolypeptide of the present disclosure.

The term “microorganism including a polypeptide variant” or“microorganism including an adenylosuccinate synthetase variant”, asused herein, refers to a microorganism provided with IMP productivity orXMP productivity in a microorganism, which naturally has a weak IMPproductivity or its parent strain has no IMP productivity or XMPproductivity. Specifically, the microorganism may be a microorganismexpressing an adenylosuccinate synthetase variant including at least oneamino acid variation in the amino acid sequence of SEQ ID NO: 2, and theamino acid modification may include the substitution of the 85^(th)amino acid from the N-terminus of the amino acid sequence of SEQ ID NO:2 with a different amino acid. Additionally, the microorganism may be amicroorganism that expresses a polypeptide variant having theadenylosuccinate synthetase activity, where the 85^(th) amino acid inthe amino acid sequence of SEQ ID NO: 2 is substituted with a differentamino acid, but the microorganism is not limited thereto.

The microorganism may be a cell or microorganism which contains apolynucleotide encoding an adenylosuccinate synthetase variant, or acell or microorganism which is transformed with a vector and is able toexpress an adenylosuccinate synthetase variant. For the purpose of thepresent disclosure, the host cell or microorganism may be anymicroorganism that can express purine nucleotides by containing theadenylosuccinate synthetase variant.

In the present disclosure, the term “microorganism producing purinenucleotides” may be used interchangeably with “purinenucleotide-producing microorganism” and “microorganism having purinenucleotide productivity”.

For the purpose of the present disclosure, the term “purine nucleotide”refers to a nucleotide including a purine-based structure, for example,IMP or XMP, but the purine nucleotide is not limited thereto.

In the present disclosure, the term “microorganism producing purinenucleotides” may be a microorganism where a genetic modification hasoccurred or activity has been enhanced for the desired purine nucleotideproduction, including both a wild-type microorganism or microorganismswhere a natural or artificial genetic modification has occurred, and themicroorganism may be a microorganism where a particular mechanism isenhanced or weakened due to reasons such as insertion of an exogenousgene, enhancement or inactivation of activity of an endogenous gene,etc. For the purpose of the present disclosure, the microorganismproducing purine nucleotides is characterized in that it has increasedproductivity of the desired purine nucleotides by containing theadenylosuccinate synthetase variant, and specifically, the microorganismmay be a microorganism of the genus Corynebacterium. Specifically, themicroorganism producing purine nucleotides or microorganism havingpurine nucleotide productivity may be a microorganism where part of thegene involved in the purine nucleotide biosynthesis pathway is enhancedor weakened, or part of the gene involved in the purine nucleotidedegradation pathway is enhanced or weakened. For example, themicroorganism may be a microorganism where expression of purf encodingphosphoribosylpyrophosphate amidotransferase is enhanced or expressionof guaB encoding inosine-5′-monophosphate dehydrogenase corresponding toIMP degradation pathway is weakened, but the microorganism is notlimited thereto.

As used herein, the term “microorganism of the genus Corynebacteriumproducing 5′-purine nucleotides” refers to a microorganism of the genusCorynebacterium which has purine nucleotide productivity naturally or bymodification. Specifically, as used herein, the microorganism of thegenus Corynebacterium having purine nucleotide productivity may be amicroorganism of the genus Corynebacterium which has improved purinenucleotide productivity by enhancing or weakening the activity of thepurA gene encoding adenylosuccinate synthetase. More specifically, asused herein, the microorganism of the genus Corynebacterium havingpurine nucleotide productivity may be a microorganism of the genusCorynebacterium which has improved purine nucleotide productivity byincluding the adenylosuccinate synthetase variant of the presentdisclosure or the polynucleotide encoding the same, or by beingtransformed with a vector including the polynucleotide encoding theadenylosuccinate synthetase variant. The “microorganism of the genusCorynebacterium having improved purine nucleotide productivity” refersto a microorganism having improved purine nucleotide productivity,compared to its parent strain before transformation or a non-variantmicroorganism. The “non-variant microorganism” refers to a wild-typestrain itself, a microorganism that does not include the protein variantproducing purine nucleotides, or a microorganism that is not transformedwith the vector containing the polynucleotide encoding theadenylosuccinate synthetase variant.

As used herein, the “microorganism of the genus Corynebacterium” may bespecifically Corynebacterium glutamicum, Corynebacterium ammoniagenes,Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacteriumthermoaminogenes, Corynebacterium efficiens, Corynebacterium stationis,etc., but the microorganism is not limited thereto.

Still another aspect of the present disclosure provides a method ofpreparing purine nucleotides, which includes culturing the microorganismof the genus Corynebacterium that produces purine nucleotide in amedium, and recovering the purine nucleotides from the microorganism orthe medium.

In the above method, culturing the microorganism may be performed by aknown batch culture, continuous culture, fed-batch culture, etc., butthe method of cultivation is not particularly limited thereto. Inparticular, the culture conditions may not be particularly limited, butan optimal pH (e.g., pH 5 to pH 9, specifically pH 6 to pH 8, and mostspecifically pH 6.8) may be adjusted using a basic compound (e.g.,sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound(e.g., phosphoric acid or sulfuric acid), and an aerobic condition maybe maintained by adding oxygen or oxygen-containing gas mixture to theculture. The culture temperature may be maintained at 20° C. to 45° C.,and specifically at 25° C. to 40° C., and the cultivation may beperformed for about 10 hours to about 160 hours, but the conditions arenot limited thereto. 5′-Inosinic acid produced by the cultivation may besecreted into the medium or may remain within the cells.

Furthermore, in the culture medium to be used, as a carbon source,sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose,maltose, molasses, starch, and cellulose), oils and fats (e.g., soybeanoil, sunflower seed oil, peanut oil, and coconut oil), fatty acids(e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g.,glycerol and ethanol), organic acids (e.g., acetic acid), etc. may beused alone or in combination, but the carbon source is not limitedthereto. As a nitrogen source, a nitrogen-containing organic compound(e.g., peptone, yeast extract, meat extract, malt extract, corn steepliquor, soybean flour, and urea) or an inorganic compound (e.g.,ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate, and ammonium nitrate), etc. may be used alone or incombination, but the nitrogen source is not limited thereto. As aphosphorus source, potassium dihydrogen phosphate, dipotassium hydrogenphosphate, a sodium-containing salt corresponding thereto, etc. may beused alone or in combination, but the phosphorus source is not limitedthereto. Additionally, the medium may also include essentialgrowth-promoting materials such as other metal salts (e.g., magnesiumsulfate or iron sulfate), amino acids, and vitamins.

A method of recovering purine nucleotides produced in the cultivationstep of the present disclosure is to collect the desired purinenucleotides from the culture using an appropriate method known in theart according to the cultivation method. For example, centrifugation,filtration, anion exchange chromatography, crystallization, HPLC, etc.may be used, and the desired purine nucleotides may be recovered fromthe medium or microorganism using an appropriate method known in theart.

Additionally, the recovering step may include a purification process.The purification process may be performed using an appropriate methodknown in the art. Therefore, the recovered purine nucleotides may be ina purified form or a microbial fermentation liquid including purinenucleotides (Introduction to Biotechnology and Genetic Engineering, A.J. Nair, 2008).

Advantageous Effects of the Invention

When a microorganism of the genus Corynebacterium producing purinenucleotides using the adenylosuccinate synthetase variant of the presentdisclosure, it is possible to produce purine nucleotides in high yield.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present disclosure will be described in detail throughexemplary embodiments. However, it will be apparent to those skilled inthe art to which the present disclosure belongs that these exemplaryembodiments are provided for the purpose of illustration only and arenot intended to limit the scope of the present disclosure.

Example 1: Preparation of Wild-Type Based IMP-Producing Strain

The wild-type strain of the genus Corynebacterium cannot produce IMP atall or can produce only a very small amount even if it is possible.Accordingly, an IMP-producing strain was prepared based onCorynebacterium stationis ATCC6872. More specifically, the IMP-producingstrain was prepared by enhancing the activity of purF gene encodingphosphoribosylpyrophosphate amidotransferase, which is the first enzymeof purine biosynthesis, and weakening the activity of guaB gene encoding5′-inosinic acid dehydrogenase that corresponds to the IMP degradationpathway.

Example 1-1: Preparation of purF-Enhanced Strain

To prepare a strain in which the start codon of purF gene is modified,an insertion vector containing the purF gene of SEQ ID NO: 3 wasprepared. To clone the purF gene into an insertion vector, specifically,PCR was performed using the genomic DNA of Corynebacterium stationisATCC6872 as a template and primers of SEQ ID NOS: 4 and 5 and SEQ IDNOS: 6 and 7 for 30 cycles of denaturation at 94° C. for 30 sec,annealing at 55° C. for 30 sec, and extension at 72° C. for 2 min. PCRwas performed again using two DNA fragments obtained by the above PCR asa template and primers of SEQ ID NOS: 4 and 72 for 30 cycles ofdenaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, andextension at 72° C. for 2 min to obtain DNA fragments. The obtained DNAfragments were digested with a restriction enzyme XbaI, and cloned intothe pDZ vector (Korean Patent No. 10-0924065 and InternationalPublication No. 2008-033001) digested with the same enzyme. Thethus-prepared vector was named as pDZ-purF-g1a.

TABLE 1 SEQ ID NO Primer Sequence (5′-3′) 4 purF gla-1GCTCTAGACCACTCTAAGACGCGGCCACC 5 purF gla-2 AAGTAGTGTTCACCATGACGCTGATTCTACTAAGTTT 6 purF gla-3 AGTAGAATCAGCGTCATGGTGAACACTAC TTTCCCCAG 7purF gla-4 GCTCTAGACTGTGCGCCCACGATATCCAG

The recombinant vector pDZ-purF-g1a was transformed into Corynebacteriumstationis ATCC6872 by electroporation, and strains in which the vectorwas inserted into the genomic DNA by homologous recombination wereselected on a medium containing 25 mg/L kanamycin. The selected primarystrains were subjected to secondary crossover, and these selectedstrains were subjected to sequencing, and thereby the desired straininto which the mutation was introduced was selected. The strain wasnamed as ATCC6872::purF(g1a) strain.

Example 1-2: Preparation of guaB-Weakened Strain

To prepare a strain in which the start codon of guaB gene is modified,an insertion vector containing the guaB gene of SEQ ID NO: 8 wasprepared. To clone the guaB gene into the insertion vector,specifically, PCR was performed using the genomic DNA of Corynebacteriumstationis ATCC6872 as a template and primers of SEQ ID NOS: 9 and 10 andSEQ ID NOS: 11 and 12. The PCR products were cloned as in Example 1-1,and the vector prepared was named as pDZ-guaB-a1t. The vector wasintroduced into the ATCC6872::purF(g1a) and the strain in which theabove mutation was introduced was finally selected.

The finally selected wild-type Corynebacterium stationis ATCC6872-basedstrain producing IMP was named as CJI2330.

TABLE 2 SEQ ID NO Primer Sequence (5′-3′)  9 guaB alt-1GCTCTAGACTACGACAACACGGTGCCTAA 10 guaB alt-2CACGATTTTCGGTCAATACGGGTCTTCTC CTTCGCAC 11 guaB alt-3AGGAGAAGACCCGTATTGACCGAAAATCG TGTTTCT 12 guaB alt-4GCTCTAGAATCGACAAGCAAGCCTGCACG

Example 1-3: Fermentation Titer Test of CJI2330

After dispensing a seed culture medium (2 mL) into test tubes (diameter:18 mm), the tubes were autoclaved. Each of ATCC6872 and CJI2330 wasinoculated and incubated at 30° C. for 24 h with shaking and used as aseed culture. A fermentation medium (29 mL) was dispensed into each 250mL shaking Erlenmeyer flask, and autoclaved at 121° C. for 15 min. Theseed culture (2 mL) was inoculated to the medium and cultured for 3days. Culture conditions were adjusted to 170 rpm, 30° C., and pH 7.5.

After completion of the culture, the amount of IMP production wasmeasured by HPLC (SHIMAZDU LC20A), and the culture results are as inTable 3 below. The following results suggest that the purf-enhanced andguaB-weakened strain has IMP productivity.

Strain IMP (g/L) ATCC6872 0 CJI2330 0.50

-   -   Seed culture medium: 1% glucose, 1% peptone, 1% meat extract, 1%        yeast extract, 0.25% sodium chloride, 100 mg/L adenine, 100 mg/L        guanine, pH 7.5    -   Fermentation medium: 0.1% sodium glutamate, 1% ammonium        chloride, 1.2% magnesium sulfate, 0.01% calcium chloride, 20        mg/L iron sulfate, 20 mg/L manganese sulfate, 20 mg/L zinc        sulfate, 5 mg/L copper sulfate, 23 mg/L L-cysteine, 24 mg/L        alanine, 8 mg/L nicotinic acid, 45 μg/L biotin, 5 mg/L thiamine        hydrochloride, 30 mg/L adenine, 1.9% phosphoric acid (85%),        2.55% glucose, 1.45% fructose

Example 2: Preparation of Adenylosuccinate Synthetase-Weakened Variant

To discover an adenylosuccinate synthetase variant capable improvingpurine nucleotide productivity, a mutant library of purA gene encodingadenylosuccinate synthetase was prepared.

Example 2-1: Preparation of Vector Containing purA Gene

To prepare a mutant library of purA gene, a recombinant vectorcontaining the purA gene was first prepared. PCR was performed using thegenomic DNA of Corynebacterium stationis ATCC6872 as a template andprimers of SEQ ID NO: 13 and SEQ ID NO: 14, and the PCR product wascloned into E. coli vector pCR2.1 using a TOPO Cloning Kit (Invitrogen)to obtain pCR-purA.

TABLE 4 SEQ ID  NO Primer Sequence (5′-3′) 13 purA 5′ primer FATGGCTAAATACATTATCACT (temp) 14 purA 3′ primer R TGTGCTGGAGACCCCTCATAG(temp)

Example 2-2: Preparation of Mutant Library of purA Gene

A mutant library of purA gene was prepared based on the vector preparedin Example 2-1. The library was prepared using an error-prone PCR kit(Clontech Diversify® PCR Random Mutagenesis Kit). Under conditions wheremutations may occur, PCR was performed using primers of SEQ ID NO: 15and SEQ ID NO: 16. Specifically, under conditions where 0 to 3 mutationsper 1000 bp may occur, pre-heating was performed at 94° C. for 30 sec,followed by 25 cycles of 94° C. for 30 sec and 68° C. for 1 min 30 sec.A PCR product thus obtained was subjected to PCR using a megaprimer (500ng to 125 ng) for 25 cycles of 95° C. for 50 sec, 60° C. for 50 sec, and68° C. for 12 min, treated with DpnI, and transformed into E. coli DH5αand spread on an LB solid medium containing kanamycin (25 mg/L). Afterselecting 20 different kinds of transformed colonies, plasmids wereobtained therefrom and subjected to sequencing analysis. As a result, itwas confirmed that mutations were introduced at different sites at afrequency of 2 mutations/kb. About 20,000 transformed E. coli colonieswere collected and the plasmids were extracted, and named as apTOPO-purA-library.

TABLE 5 SEQ ID  NO Primer Sequence (5′-3′) 15 purA error PCRAAGGGCAAAGCTACAGACATC primer F 16 purA error PCR CCGCCGAGCATGAGAACCCGAprimer R

Example 3: Evaluation of Prepared Library and Selection of StrainExample 3-1: Evaluation of Library

The pTOPO-purA-library prepared in Example 2-2 was transformed into theCJI2330 strain prepared in Example 1 by electroporation, and the strainwas spread on a nutrient medium containing 25 mg/L kanamycin to obtain10,000 colonies into which the mutant gene was inserted. Each of thecolonies was named as CJI2330::pTOPO_purA(mt)1 toCJI2330::pTOPO_purA(mt)10000.

-   -   Nutrient medium: 1% peptone, 1% meat extract, 0.25% sodium        chloride, 1% yeast extract, 2% agar, pH 7.2

Each of the obtained 10,000 colonies was inoculated in 200 μL of anautoclaved seed culture medium, and cultured in a 96-deep well platewith shaking at 30° C., 1200 rpm for 24 hours using a microplate shaker(TAITEC), and used as a seed culture. The autoclaved fermentation medium(290 μL) was dispensed into a 96-deep well plate, and 20 μL of each ofthe seed cultures was inoculated thereto, followed by culturing withshaking under the same conditions as above for 72 hours.

To analyze the 5′-inosinic acid produced in the culture medium, aftercompletion of the culture, 3 μL of the culture supernatant wastransferred to a 96-well UV-plate, where each well contained 197 μL ofdistilled water, and shaken for 30 sec using a microplate reader, andabsorbance was measured 270 nm at 25° C. using a spectrophotometer. Theabsorbance was compared with that of the CJI2330 strain, and 50 coloniesof mutant strains showing a 10% or more increase in the absorbance wereselected. Other colonies showed similar or decreased absorbance comparedto the control.

The absorbance of the 50 selected strains was measured in the samemanner as above to repeatedly examine the amount of 5′-inosinic acidproduction. One strain, CJI2330::pTOPO_purA(mt)333, which showed asignificant improvement in 5′-inosinic acid productivity compared to theCJI2330 strain, was selected.

To confirm the validity of selected mutants, a fermentation titer testwas performed.

After dispensing a seed culture medium (2 mL) into test tubes (diameter:18 mm), the tubes were autoclaved. Each of CJI2330 andCJI2330::pTOPO_purA(mt)333 was inoculated and incubated at 30° C. for 24h with shaking and used as a seed culture. A fermentation medium (29 mL)was dispensed into each 250 mL shaking Erlenmeyer flask, and autoclavedat 121° C. for 15 min. The seed culture (2 mL) was inoculated to themedium and cultured for 3 days. Culture conditions were adjusted to 170rpm, 30° C., and pH 7.5.

After completion of the culture, the amount of IMP production wasmeasured by HPLC (SHIMAZDU LC20A), and the culture results are as inTable 6 below.

TABLE 6 Strain IMP (g/L) CJI2330 0.50 CJI2330::pTOPO_purA(mt)333 0.61

As can be seen from the above results, it was confirmed that the amountof IMP was increased by about 122% in the strain where a vectorcontaining a purA gene mutation compared to the CJI2330 strain.Accordingly, it was determined that the selected mutation in the librarywas valid.

Example 3-2: Confirmation of purA Variation

To confirm the gene variation of the mutant strain, PCR was performed inthe CJI2330::pTOPO_purA(mt)333 strain using primers of SEQ ID NOS: 17and 18, and the PCR product was subjected to sequencing, therebyconfirming the presence of variation in the purA gene.

TABLE 7 SEQ ID NO Primer Sequence (5′-3′) 17 purA seq FGACGCGTCGGAATCGCCGATA 18 purA seq R CCGCCGAGCATGAGAACCCGA

Specifically, it was confirmed that the purA gene of theCJI2330::pTOPO_purA(mt)333 strain includes a variation where the 85^(th)amino acid (i.e., glycine) of the purA amino acid sequence representedby SEQ ID NO: 2 is substituted with serine (i.e., the 253^(rd)nucleotide, ‘g’, is substituted with a nucleotide ‘a’). Accordingly, inExamples hereinbelow, attempts were made to confirm whether the abovevariation can affect the amount of purine nucleotide production in eachmicroorganism of the genus Corynebacterium.

Example 4: Confirmation of IMP Production in IMP-Producing StrainDerived from ATCC6872

An IMP-producing strain derived from ATCC6872 was prepared, and thevariation confirmed in Example 3 was introduced into the strain and theIMP productivity of the strain was confirmed.

Example 4-1: Selection of IMP-Producing Strain Derived From ATCC6872

To prepare an IMP-producing strain derived from the ATCC6872 strain, theculture of ATCC6872 was suspended in a phosphate buffer (pH 7.0) orcitrate buffer (pH 5.5) at a density of 10⁷ cells/mL to 10⁸ cells/mL andtreated with UV at room temperature or 32° C. for 20 min to 40 min toinduce a mutation. The strain was washed with a 0.85% saline solutiontwice and spread, after dilution, on a minimal medium containing 1.7%agar which was supplemented with a resistance-providing material at anappropriate concentration, and thereby colonies were obtained. Eachcolony was cultured in a nutrient medium and then cultured in a seedculture medium for 24 hours. After culturing each colony in afermentation medium for 3 to 4 days, colonies which showed excellentproduction of IMP accumulated in the culture medium were selected. Toprepare a strain producing IMP at high concentration, adenine-auxotroph,guanine-leaky type, lysozyme sensitivity, 3,4-dehydroproline resistance,streptomycin resistance, sulfaguanidine resistance, norvalineresistance, and trimethoprim resistance were provided by performing thecorresponding procedures in a sequential manner. As a result, theCJI12335 strain provided with resistance to the above materials andhaving excellent IMP productivity was finally selected. The resistancesof the CJI2332 strain relative to those of ATCC6872 were compared andthe results are shown in the following Table 8.

TABLE 8 Characteristic ATCC6872 CJI2332 Adenine-auxotroph Non-auxotrophAuxotroph Guanine-leaky type Non-auxotroph Leaky type Lysozymesensitivity 80 μg/mL  8 μg/mL 3,4-Dehydroproline 1000 μg/mL  3,500μg/mL   resistance Streptomycin resistance 500 μg/mL  2,000 μg/mL  Sulfaguanidine resistance 50 μg/mL 200 μg/mL Norvaline resistance 0.2mg/mL   2 mg/mL Trimethoprim resistance 20 μg/mL 100 μg/mL

-   -   Minimal medium: 2% glucose, 0.3% sodium sulfate, 0.1%        monopotassium phosphate, 0.3% dipotassium phosphate, 0.3%        magnesium sulfate, 10 mg/L calcium chloride, 10 mg/L iron        sulfate, 1 mg/L zinc sulfate, 3.6 mg/L manganese chloride, 20        mg/L L-cysteine, 10 mg/L calcium pantothenate, 5 mg/L thiamine        hydrochloride, 30 μg/L biotin, 20 mg/L adenine, 20 mg/L guanine,        adjusted to pH 7.3.

Example 4-2: Fermentation Titer Test of CJI2332

After dispensing a seed culture medium (2 mL) into test tubes (diameter:18 mm), the tubes were autoclaved. Each of ATCC6872 and CJI2332 wasinoculated and incubated at 30° C. for 24 hours with shaking and used asa seed culture. A fermentation medium (29 mL) was dispensed into each250 mL shaking Erlenmeyer flask, and autoclaved at 121° C. for 15 min.The seed culture (2 mL) was inoculated to the medium and cultured for 3days. Culture conditions were adjusted to 170 rpm, 30° C., and pH 7.5.

After completion of the culture, the amount of IMP production wasmeasured by HPLC (SHIMAZDU LC20A), and the culture results are as inTable 9 below.

TABLE 9 Strain IMP (g/L) ATCC6872 0 CJI2332 1.74

Example 4-3: Preparation of Insertion Vector Containing purA Variation

To introduce the variations selected in Example 3 into the strains, aninsertion vector was prepared. The process for preparing the vector forintroduction of purA(G85S) variation is as follows. PCR was performedusing the CJI2330::Topo_purA(G85S) as a template and primers of SEQ IDNO: 55 and SEQ ID NO: 56. PCR was performed as follows: denaturation at94° C. for 5 min; 20 cycles of denaturation at 94° C. for 30 sec,annealing at 55° C. for 30 sec, and polymerization at 72° C. for 1 min;and polymerization at 72° C. for 5 min. The thus obtained gene fragmentswere each digested with XbaI. Each gene fragment was cloned into alinear pDZ vector digested with XbaI using T4 ligase, and thereby thepDZ-purA(G85S) vector was prepared.

TABLE 10 SEQ ID NO Primer Sequence (5′-3′) 55 purA(G855) F′GCTCTAGATGCCGGCATTTTTCGAAGC 56 purA(G85S) RGCTCTAGAAAGTAGTCGGTAAAGCCGTTG

Example 4-4: Introduction of Variants into CJI2330 and CJI2332 StrainsDerived from ATCC6872 and their Evaluation

The purA variation was introduced to each of the wild-type-derivedIMP-producing CJI2330 strain prepared in Example 1 and the CJI2332strain selected in Example 4-1, and the amount of IMP produced by eachstrain was evaluated. To confirm the presence of a variation in the purAgene, the chromosomal DNA of the CJI2332 strain was amplified by PCR.Specifically, first, purA gene fragments were amplified by PCR using thechromosomal DNA of the CJI2332 strain as a template and primers of SEQID NOS: 17 and 18, in which the PCR was performed by 28 cycles ofdenaturation at 94° C. for 1 min; annealing at 58° C. for 30 sec, andpolymerization at 72° C. for 2 min using Taq DNA polymerase. Thenucleotide sequences of the amplified purA fragments were analyzed usingthe same primers, and as a result, it was confirmed that there was novariation in the purA gene of the CJI2332 strain.

Then, the pDZ-purA(G85S) vector was transformed into the CJI2330 strainand the CJI2332 strain, and the strains in which the vector was insertedon the genomic DNA by recombination of homologous sequences wereselected on a medium containing kanamycin (25 mg/L). The selectedprimary strains were subjected to secondary crossover, and thereby thestrains in which a variation of the target gene was introduced wereselected. For confirmation of the introduction of the gene variation inthe desired transformed strains, PCR was performed using primers of SEQID NO: 17 and SEQ ID NO: 18 and the PCR products were confirmed bysequence analysis. As a result, it was confirmed that the gene variationwas introduced into the strains. The thus-prepared strains were named asCJI2330::purA(G85S) and CJI2332::purA(G85S), respectively.

The IMP productivity for each of CJI2330, CJI2332, CJI2330::purA(G85S),and CJI2332::purA(G85S) strains was evaluated. After the completion ofculture, the amount of IMP production by each strain was measured by amethod using HPLC, and the culture results are shown in Table 11 below.

TABLE 11 Strain IMP (g/L) CJI2330 0.50 CJI2330::purA(G85S) 0.61 CJI23321.74 CJI2332::purA(G85S) 2.03

In the above results, it was confirmed that the strain in which the purAgene variation was introduced showed an increase in the amount of IMPproduction by about 122% and about 116% compared to thewild-type-derived IMP-producing CJI2330 and CJI2332 strains,respectively.

The CJI2332 strain was deposited at the Korean Culture Center ofMicroorganisms (KCCM) on Jun. 22, 2018, under the provisions of theBudapest Treaty and assigned accession number KCCM12277P. Additionally,the prepared CJI2332::purA(G85S) strain, also called CJI2348, wasdeposited at the KCCM on Jun. 22, 2018, under the provisions of theBudapest Treaty and assigned accession number KCCM12280P.

Example 5: Confirmation of 5′-Xanthylic Acid Productivity by purA GeneVariation Example 5-1: Selection of XMP-Producing Strains Derived fromATCC6872

To prepare a 5′-xanthosine monophosphate (XMP)-producing strain derivedfrom ATCC6872, the Corynebacterium stationis ATCC6872 strain wassuspended in the phosphate buffer (pH 7.0) or citrate buffer (pH 5.5) ata density of 10⁷ cells/mL to 10⁸ cells/mL and treated with UV at roomtemperature or 32° C. for 20 min to 40 min to induce a mutation. Thestrain was washed with a 0.85% saline solution twice and spread, afterdilution, on a minimal medium containing 1.7% agar which wassupplemented with a resistance-providing material at an appropriateconcentration, and thereby colonies were obtained. Each colony wascultured in a nutrient medium and then cultured in a seed culture mediumfor 24 h. After culturing each colony in a fermentation medium for 3 to4 days, colonies which showed excellent production of XMP accumulated inthe culture medium were selected. Specifically, strains were selectedfrom those which can grow in a medium where fluorotryptophan is addedaccording to concentrations (addition medium), and more specifically,from those which can grow in a medium with a fluorotryptophanconcentration of 100 mg/L and has an improved concentration of5′-xanthylic acid. The selected strain was named as CJX1664.

-   -   Minimal medium: glucose 20 g/L, monopotassium phosphate 1 g/L,        dipotassium phosphate 1 g/L, urea 2 g/L, ammonium sulfate 3 g/L,        magnesium sulfate 1 g/L, calcium chloride 100 mg/L, iron sulfate        20 mg/L, manganese sulfate 10 mg/L, zinc sulfate 10 mg/L, biotin        30 μg/L, thiamine hydrochloride 0.1 mg/L, copper sulfate 0.8        mg/L, adenine 20 mg/L, guanine 20 mg/L, pH 7.2    -   Addition medium: a medium where fluorotryptophan at a        concentration of 10 mg/L, 20 mg/L, 50 mg/L, 70 mg/L, 100 mg/L,        and 200 mg/L is added to a minimal medium

The biochemical characteristics of the CJX1664 strain are shown in Table12 below. Referring to Table 12, the CJX1664 strain can be grown in anaddition medium where a fluorotryptophan is added at a concentration of100 mg/L.

TABLE 12 Characteristics ATCC6872 CJX1664 Fluorotryptophan Resistance 10mg/L 100 mg/L

Example 5-2: CJX1664 Fermentation Titer Test

After dispensing a seed culture medium (2 mL) into test tubes (diameter:18 mm), the tubes were autoclaved. Each of ATCC6872 and CJX1664 wasinoculated and incubated at 30° C. for 24 h with shaking and used as aseed culture. A fermentation medium (29 mL) was dispensed into each 250mL shaking Erlenmeyer flask, and autoclaved at 121° C. for 15 min. Theseed culture (2 mL) was inoculated to the medium and cultured for 3days. Culture conditions were adjusted to 170 rpm, 30° C., and pH 7.5.

After completion of the culture, the amount of XMP production wasmeasured by HPLC (SHIMAZDU LC20A), and the culture results are as inTable 13 below.

TABLE 13 Strain XMP (g/L) ATCC6872 0 CJX1664 4.72

Example 5-3: Introduction of Variant into CJX1664 Strain and theirEvaluation

To confirm the presence of a variation of the purA gene of the CJX1664strain selected in Example 5-1, the chromosomal DNA PCR of the CJX1664strain was amplified by PCR. Specifically, first, purA fragments wereamplified by PCR using the chromosomal DNA of the CJX1664 strain as atemplate and primers of SEQ ID NOS: 17 and 18, in which the PCR wasperformed by 28 cycles of denaturation at 94° C. for 1 min; annealing at58° C. for 30 sec, and polymerization at 72° C. for 2 min using Taq DNApolymerase. The nucleotide sequences of the amplified purA genefragments were analyzed using the same primers, and as a result, it wasconfirmed that there was no variation in the purA gene of the CJX1664strain.

The vector prepared in Example 4-3 was transformed into the CJX1664strain, and the strains in which the vector was inserted on the genomicDNA by recombination of homologous sequences were selected on a mediumcontaining 25 mg/L kanamycin. The selected primary strains weresubjected to secondary crossover, and thereby those strains in which avariation of the target gene was introduced were selected. Theintroduction of the gene variation in the desired transformed strainswas confirmed by sequence analysis.

The XMP productivity for each of CJX1664 and CJX1664::purA(G85S) strainswas evaluated. After the completion of culture, the amount of XMPproduction by each strain was measured by a method using HPLC, and theculture results are shown in Table 14 below.

TABLE 14 Strain XMP (g/L) CJX1664 4.72 CJX1664::purA(G85S) 5.19

As can be seen in Table 14 above, the CJX1664::purA(G85S) strain showedan increase in the amount of XMP production by about 109% compared tothe CJX1664 strain (i.e., an ATCC6872-based XMP-producing strain).

The CJX1664 strain was deposited at the Korean Culture Center ofMicroorganisms (KCCM) on Jul. 6, 2018, under the provisions of theBudapest Treaty and assigned accession number KCCM12285P. Additionally,the prepared CJX1664::purA(G85S) strain, also called CJX1665, wasdeposited at the KCCM on Jul. 6, 2018, under the provisions of theBudapest Treaty and assigned accession number KCCM12286P.

Example 6: Substitution of Amino Acid in purA Variation with DifferentAmino Acid Example 6-1: Preparation of Vector for Insertion of AminoAcid in purA Variation

Through the above Examples, it was confirmed that the purA(G85S)variation can improved the productivity of purine nucleotides. In thisregard, to confirm the positional importance of the purA variation, theeffect of the substitution of the 85^(th) amino acid with a differentamino acid on the productivity of purine nucleotides was examined. Theprocess of preparing the vector for the insertion of purA(G85S)variation is as follows. Site-directed mutagenesis was performed usingthe pDZ-purA(G85S) vector prepared in Example 4 as a backbone.Specifically, PCR was performed using the sequences shown in Table 15 asprimers under the following conditions: 18 cycles of denaturation at 94°C. for 30 sec, annealing at 55° C. for 30 sec, and extension at 68° C.for 12 min. The resulting PCR products were digested with DpnI,transformed into a DH5α strain, and colonies were obtained therefrom.The plasmids of thus obtained colonies were obtained by a known plasmidextraction method, and the information of the obtained plasmids areshown below in Table 15.

TABLE 15 SEQ ID NO Primer Sequence (5′-3′) 21 purA(G85A) FCTTTGAGGAAATTGAAGCTCTCGAAGCCCGCGGCGC 22 purA(G85A) RGCGCCGCGGGCTTCGAGAGCTTCAATTTCCTCAAAG 23 purA(G85V) FCTTTGAGGAAATTGAAGTCCTCGAAGCCCGCGGCGC 24 purA(G85V) RGCGCCGCGGGCTTCGAGGACTTCAATTTCCTCAAAG 25 purA(G85L) FCTTTGAGGAAATTGAACTGCTCGAAGCCCGCGGCGC 26 purA(G85L) RGCGCCGCGGGCTTCGAGCAGTTCAATTTCCTCAAAG 27 purA(G85M) FCTTTGAGGAAATTGAAATGCTCGAAGCCCGCGGCGC 28 purA(G85M) RGCGCCGCGGGCTTCGAGCATTTCAATTTCCTCAAAG 29 purA(G85I) FCTTTGAGGAAATTGAAATCCTCGAAGCCCGCGGCGC 30 purA(G85I) RGCGCCGCGGGCTTCGAGGATTTCAATTTCCTCAAAG 31 purA(G85T) FCTTTGAGGAAATTGAAACTCTCGAAGCCCGCGGCGC 32 purA(G85T) RGCGCCGCGGGCTTCGAGAGTTTCAATTTCCTCAAAG 33 purA(G85N) FCTTTGAGGAAATTGAAAACCTCGAAGCCCGCGGCGC 34 purA(G85N) RGCGCCGCGGGCTTCGAGGTTTTCAATTTCCTCAAAG 35 purA(G85Q) FCTTTGAGGAAATTGAACAGCTCGAAGCCCGCGGCGC 36 purA(G85Q) RGCGCCGCGGGCTTCGAGCTGTTCAATTTCCTCAAAG 37 purA(G85C) FCTTTGAGGAAATTGAATGCCTCGAAGCCCGCGGCGC 38 purA(G85C) RGCGCCGCGGGCTTCGAGGCATTCAATTTCCTCAAAG 39 purA(G85P) FCTTTGAGGAAATTGAACCACTCGAAGCCCGCGGCGC 40 purA(G85P) RGCGCCGCGGGCTTCGAGTGGTTCAATTTCCTCAAAG 41 purA(G85Y) FCTTTGAGGAAATTGAATACCTCGAAGCCCGCGGCGC 42 purA(G85Y) RGCGCCGCGGGCTTCGAGGTATTCAATTTCCTCAAAG 43 purA(G85W) FCTTTGAGGAAATTGAATGGCTCGAAGCCCGCGGCGC 44 purA(G85W) RGCGCCGCGGGCTTCGAGCCATTCAATTTCCTCAAAG 45 purA(G85K) FCTTTGAGGAAATTGAAAAGCTCGAAGCCCGCGGCGC 46 purA(G85K) RGCGCCGCGGGCTTCGAGCTTTTCAATTTCCTCAAAG 47 purA(G85R) FCTTTGAGGAAATTGAACGCCTCGAAGCCCGCGGCGC 48 purA(G85R) RGCGCCGCGGGCTTCGAGGCGTTCAATTTCCTCAAAG 49 purA(G85H) FCTTTGAGGAAATTGAACACCTCGAAGCCCGCGGCGC 50 purA(G85H) RGCGCCGCGGGCTTCGAGGTGTTCAATTTCCTCAAAG 51 purA(G85D) FCTTTGAGGAAATTGAAGATCTCGAAGCCCGCGGCGC 52 purA(G85D) RGCGCCGCGGGCTTCGAGATCTTCAATTTCCTCAAAG 53 purA(G85E) FCTTTGAGGAAATTGAAGAACTCGAAGCCCGCGGCGC 54 purA(G85E) RGCGCCGCGGGCTTCGAGTTCTTCAATTTCCTCAAAG

TABLE 16 No. Plasmid 1 pDZ-purA G85A 2 pDZ-purA G85V 3 pDZ-purA G85L 4pDZ-purA G85M 5 pDZ-purA G85I 6 pDZ-purA G85T 7 pDZ-purA G85N 8 pDZ-purAG85 Q 9 pDZ-purA G85C 10 pDZ-purA G85P 11 pDZ-purA G85Y 12 pDZ-purA G85W13 pDZ-purA G85K 14 pDZ-purA G85R 15 pDZ-purA G85H 16 pDZ-purA G85D 17pDZ-purA G85E

Example 6-2: Preparation of Strain where an Amino Acid is Substitutedwith a Different Amino Acid According to Position of Variation of a purAVariant, and Comparison of 5′-Inosinic Acid Productivities

Each of the 18 kinds of vectors, for the introduction of variants,prepared in Example 6-1 was transformed into the CJI2332 strain, andthose strains where these vectors were inserted into the genomic DNA byhomologous recombination were selected on a medium containing 25 mg/Lkanamycin. The selected primary strains were subjected to secondarycrossover, and thereby those strains into which a variation of thetarget gene was introduced were selected. For confirmation of theintroduction of the gene variation in the desired transformed strains,PCR was performed using primers of SEQ ID NO: 17 and SEQ ID NO: 18 andthe PCR products were confirmed by sequence analysis. The strains werenamed according to the inserted varions as shown in Table 17.

TABLE 17 No. Strain 1 CJI2332::purA(G85A) 2 CJI2332::purA(G85V) 3CJI2332::purA(G85L) 4 CJI2332::purA(G85M) 5 CJI2332::purA(G85I) 6CJI2332::purA(G85T) 7 CJI2332::purA(G85N) 8 CJI2332::purA(G85Q) 9CJI2332::purA(G85C) 10 CJI2332::purA(G85P) 11 CJI2332::purA(G85Y) 12CJI2332::purA(G85W) 13 CJI2332::purA(G85K) 14 CJI2332::purA(G85R) 15CJI2332::purA(G85H) 16 CJI2332::purA(G85D) 17 CJI2332::purA(G85E)

The concentration of 5′-inosinic acid was analyzed by culturing thestrains in the same manner as in Example 1.

TABLE 18 Concentration of 5′-inosinic acid with variation in purA (g/L)Average No. Strain 5′-Inosinic acid CJI2332 1.74 ControlCJI2332::purA(G85S) 2.03 Group 1 CJI2332::purA(G85A) 1.93 2CJI2332::purA(G85V) 1.84 3 CJI2332::purA(G85L) 2.01 4CJI2332::purA(G85M) 2.01 5 CJI2332::purA(G85I) 2.02 6CJI2332::purA(G85T) 2.02 7 CJI2332::purA(G85N) 1.83 8CJI2332::purA(G85Q) 2.03 9 CJI2332::purA(G85C) 1.82 10CJI2332::purA(G85P) 1.10 11 CJI2332::purA(G85Y) 1.92 12CJI2332::purA(G85W) 0.39 13 CJI2332::purA(G85K) 1.86 14CJI2332::purA(G85R) 1.30 15 CJI2332::purA(G85H) 1.67 16CJI2332::purA(G85D) 2.02 17 CJI2332::purA(G85E) 1.94

Referring to Table 18 above, it was confirmed that the strainscontaining the purA, in which the 85^(th) amino acid of the amino acidsequence encoding the purA gene is substituted with a different aminoacid, showed a significant change in the amount of IMP production,compared to other strains which did not contain the above variation.That is, it was confirmed that the 85^(th) amino acid of the amino acidsequence encoding the purA gene is an important position for variationassociated with the production of purine nucleotides, and when the85^(th) amino acid of the amino acid sequence encoding the purA gene issubstituted with an amino acid selected from the group consisting ofserine, alanine, valine, leucine, methionine, isoleucine, threonine,asparagine, glutamine, cysteine, tyrosine, lysine, aspartic acid, andglutamic acid, the microorganism having the variation can significantlyincrease the production of purine nucleotides.

From the foregoing, a skilled person in the art to which the presentdisclosure pertains will be able to understand that the presentdisclosure may be embodied in other specific forms without modifying thetechnical concepts or essential characteristics of the presentdisclosure. In this regard, the exemplary embodiments disclosed hereinare only for illustrative purposes and should not be construed aslimiting the scope of the present disclosure. On the contrary, thepresent disclosure is intended to cover not only the exemplaryembodiments but also various alternatives, modifications, equivalents,and other embodiments that may be included within the spirit and scopeof the present disclosure as defined by the appended claims.

The invention claimed is:
 1. A polynucleotide encoding anadenylosuccinate synthetase variant having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:2 and havingadenylosuccinate synthetase activity, wherein the amino acidcorresponding to position 85 of the amino acid sequence of SEQ ID NO: 2is substituted with a different amino acid, wherein the different aminoacid is selected from the group consisting of serine, alanine, valine,leucine, methionine, isoleucine, threonine, asparagine, glutamine,cysteine, tyrosine, lysine, aspartic acid, and glutamic acid, andwherein said adenylosuccinate synthetase variant has increased purineproduction relative to a wild type adenylosuccinate synthetase.
 2. Avector comprising the polynucleotide of claim
 1. 3. A microorganism ofthe genus Corynebacterium capable of producing purine nucleotides,comprising an adenylosuccinate synthetase variant having at least 90%sequence identity to the amino acid sequence of SEQ ID NO:2 and havingadenylosuccinate synthetase activity, wherein the amino acidcorresponding to position 85 of the amino acid sequence of SEQ ID NO: 2is substituted with a different amino acid, wherein the different aminoacid is selected from the group consisting of serine, alanine, valine,leucine, methionine, isoleucine, threonine, asparagine, glutamine,cysteine, tyrosine, lysine, aspartic acid, and glutamic acid, andwherein said adenylosuccinate synthetase variant has increased purineproduction relative to a wild type adenylosuccinate synthetase.
 4. Themicroorganism according to claim 3, wherein the microorganism of thegenus Corynebacterium is Corynebacterium stationis.
 5. A method forpreparing IMP, XMP or GMP, comprising: culturing the microorganism ofthe genus Corynebacterium of claim 3 in a medium; and recovering theIMP, XMP or GMP from the microorganism or the medium.
 6. The methodaccording to claim 5, wherein the microorganism of the genusCorynebacterium is Corynebacterium stationis.
 7. A microorganism of thegenus Corynebacterium capable of producing purine nucleotides,comprising the vector of claim
 2. 8. The microorganism according toclaim 7, wherein the microorganism of the genus Corynebacterium isCorynebacterium stationis.
 9. A method for preparing IMP, XMP or GMP,comprising: culturing the microorganism of the genus Corynebacterium ofclaim 7 in a medium; and recovering the IMP, XMP or GMP from themicroorganism or the medium.
 10. The method according to claim 9,wherein the microorganism of the genus Corynebacterium isCorynebacterium stationis.